siRNA resistant gene - How many bp to change? (Mar/12/2009 )

Hi everyone!

I am new to siRNA and I was told that the "gold standard" for assuring you are observing gene-specific alterations is to create a siRNA resistant form and transfect that in to rescue the phenotype. I have found that the best amino acid codons to mutate are ser, arg, and leu due to the redundancy in these, but I was wondering if anyone knows how many bp mutations are ideal? It seems like mutating 1-2bp would be insufficient?

Any resources you could point me towards would also be appreciated!!! THANKS!

-LabGirl-

Do you mean mutate the siRNA sequence? You can mutate the "seed" sequence of the siRNA which is the #2-#8 nt counted from the 5' end of the antisense strand. I think 4 nt mutation should be sufficient in disrupting RNAi activity. But make sure the mutated sequence does not create new targets.

-pcrman-

pcrman on Mar 12 2009, 05:34 PM said:

Do you mean mutate the siRNA sequence? You can mutate the "seed" sequence of the siRNA which is the #2-#8 nt counted from the 5' end of the antisense strand. I think 4 nt mutation should be sufficient in disrupting RNAi activity. But make sure the mutated sequence does not create new targets.

I think LabGirl want to generate a RNAi resistant gene which contains silent mutations and is thus not recognized by the siRNA.

I had done this before. I changed every changeable bp.

-WHR-

WHR on Mar 13 2009, 03:25 AM said:

pcrman on Mar 12 2009, 05:34 PM said:

Do you mean mutate the siRNA sequence? You can mutate the "seed" sequence of the siRNA which is the #2-#8 nt counted from the 5' end of the antisense strand. I think 4 nt mutation should be sufficient in disrupting RNAi activity. But make sure the mutated sequence does not create new targets.

I think LabGirl want to generate a RNAi resistant gene which contains silent mutations and is thus not recognized by the siRNA.

I had done this before. I changed every changeable bp.

Yes, WHR was right-I meant creating a resistant gene with silent mutations that won't be recognized by the siRNA-I just wasn't sure how many bp to mutate to ensure the siRNA doesn't recognize it.

-LabGirl-

LabGirl on Mar 13 2009, 07:25 AM said:

Yes, WHR was right-I meant creating a resistant gene with silent mutations that won't be recognized by the siRNA-I just wasn't sure how many bp to mutate to ensure the siRNA doesn't recognize it.

The specificity study in this paper might prove useful. They mapped the effect of mispairs at various positions of the Argonaute-bound RNA.

The work was based on a Thermus thermophilus argonaute. Therefore the results may not precisely describe the behavior of argonaute in your system. However, the specificity study is good and is worth looking over.